The structure of a polypeptide is an important element in its activity. Oftentimes, a properly folded polypeptide needs to associate with itself or different polypeptides in order to be active. Most often these interactions are as homodimers and provide an active complex, however, heterotrimers also occur. Defined trimerizing motifs are typically coiled coil domains (Burkhard et al., 2001, Trends in Cell Biol., 11:82), and includes SP-D trimerizing domains (U.S. Pat. Nos. 5,716,805 and 6,190,886).
Although lacking a distinctive coiled coil motif, additional molecules known to require trimerization for optimal activity include the tumor necrosis factor super family, such as TNF-alpha, lymphotoxin alpha, and CD40L (TNFSF5), among others. Without trimerization, many TNF super family polypeptides have low or absent activity (Fanslow et al., (1994) Seminars Immunology, 6:267–278). CD40 is a transmembrane polypeptide member of the tumor necrosis factor receptor super family and is involved in stimulating proliferation and differentiation of humoral and cellular immune cells. In particular, CD40 is involved in isotype switching and is important in T-cell activation and production of type 1 cytokines in response to protein antigens (Noelle, R, (1996) Immunity, 4:415; and Borrow et al., (1996) J. Exp. Med., 183:2129).
It has been shown that CD40L (TNFSF5) is normally active in a membrane bound form, however, this form is difficult to administer as a therapeutic. The soluble version of CD40L (TNFSF5) was found to have very little stimulatory activity, but by trimerizing soluble CD40L (TNFSF5) using a mutated GCN4 leucine zipper domain, significant activation of target cells was achieved (Fanslow et al., (1994) Seminars Immunology, 6:267–278; Morris et al., (1999) J. Biol. Chem., 274:418–423; U.S. Pat. No. 5,716,805).
A significant limitation of using the mutant leucine zipper trimerizing domain in a therapeutic fusion polypeptide is that leucine zippers are typically nuclear proteins, i.e., intracellular. Thus even though the polypeptide may be of the same species as a subject being treated with the polypeptide, having not been seen by the immune system can lead to the polypeptide being recognized as foreign when expressed extracellularly and triggering an immune response in the subject. This response is particularly deleterious to the continuous or long-term administration of the fusion polypeptide to a patient. The present invention addresses this issue by providing polypeptide domains that will have reduced immunogenicity in addition to having trimerization properties.